LAMP-LFD (loop-mediated isothermal amplification and lateral flow dipstick) detection kit and detection method for Listeria monocytogenes

A LAMP-LFD, Listeria monocytogenes technology, applied in the fields of animal health and food inspection, can solve the problems of difficult identification of similar species or subtypes, complicated bacterial culture operations, and adaptability defects, etc., to improve epidemic monitoring and rapid development. Detection level, detection convenience, and the effect of improving detection capabilities

Inactive Publication Date: 2015-01-07
天津市动物疫病预防控制中心
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

The traditional diagnosis of Listeria monocytogenes is mainly completed by pathogen isolation and biochemical identification, but it has the disadvantages of cumbersome bacterial culture operations, time-consuming, and difficult identification of similar species or subtypes.
With the development of molecular biology detection technology, PCR or real-time quantitative fluorescent PCR technology established with hlyA gene, inl gene, iap gene as the target has been successfully applied to the laboratory diagnosis of Listeria monocytogenes, which has strong specificity, It has the advantages of high sensitivity and fast speed, but this method must be equipped with expensive instruments and equipment, requires specialized operators, and has certain defects in adaptability

Method used

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  • LAMP-LFD (loop-mediated isothermal amplification and lateral flow dipstick) detection kit and detection method for Listeria monocytogenes
  • LAMP-LFD (loop-mediated isothermal amplification and lateral flow dipstick) detection kit and detection method for Listeria monocytogenes
  • LAMP-LFD (loop-mediated isothermal amplification and lateral flow dipstick) detection kit and detection method for Listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Extraction of Listeria monocytogenes DNA: Use the cell / tissue DNA extraction kit of Tiangen Biological Company, and refer to the instruction manual for the method.

[0045] (1) Take 1ml of the bacterial solution and centrifuge at 12,000rpm for 5 minutes, remove the supernatant, add 200μL GA to the pellet, resuspend and mix well.

[0046] (2) Add 20 μL proteinase K solution and mix well. The samples were mixed by inversion 2-3 times in a water bath at 65°C for 1 hour. Remove from the water bath and centrifuge briefly to remove water droplets from the inside of the cap.

[0047] (3) Add 200 μL of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 minutes. The solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0048] (4) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds. At this time, flocculent sediment may appear. Briefly centrifuge to remove water droplets on the inner wal...

Embodiment 2

[0064] 1. DNA extraction of Listeria monocytogenes in food:

[0065] The overnight cultured Listeria monocytogenes bacteria liquid (the original bacteria concentration was 3.6×10 6 cfu / ml) diluted to 10 -1 ~10 -8 , take 1ml of the bacterial solution of each dilution and add it to the commercially available pork to make a simulated test sample. The DNA in the tissue was extracted by the method of Example 1. Take 3 μL of DNA template for LAMP-LFD detection respectively.

[0066] 2. Amplification:

[0067] (1) Reagents: Bst DNA polymerase large fragment produced by BioLabs (NEW ENGLAND); LAMP reaction solution; Listeria monocytogenes LAMP primers; lateral flow test strips produced by Milenia Biotec GmbH

[0068] (2) Amplification reaction system The total volume of the amplification reaction is 25 μL, and its various components and final concentrations are: 2×Buffer (including Mg 2+ ) 2.5 μL; LM-F3 0.5 μL; LM-B3 0.5 μL; LM-FIP 0.5 μL; LM-BIP 0.5 μL;

[0069] (3) Amplificat...

Embodiment 3

[0074] 1. Specificity test of Listeria monocytogenes LAMP-LFD kit:

[0075] (1) Control strains include:

[0076]

[0077] (2) Viral DNA extraction: Use the cell / tissue DNA extraction kit from Tiangen Biological Company, and refer to the instruction manual for the method.

[0078] 2. Amplification reaction:

[0079] (1) Reagents: Bst DNA polymerase large fragment produced by BioLabs (NEW ENGLAND); LAMP reaction solution; Listeria monocytogenes LAMP primers; lateral flow test strips produced by Milenia Biotec GmbH

[0080] (2) Amplification reaction system The total volume of the amplification reaction is 25 μL, and its various components and final concentrations are: 2×Buffer (including Mg 2+ ) 2.5 μL; LM-F3 0.5 μL; LM-B3 0.5 μL; LM-FIP 0.5 μL; LM-BIP 0.5 μL;

[0081] (3) Amplification reaction process: 60min at 63°C.

[0082] (4) After the amplification reaction was completed, 20 pmol of probe LM-HP was added to the reaction system without termination reaction, and ...

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Abstract

The invention relates to a rapid detection kit and a detection method for Listeria monocytogenes. The technology can be used for detecting Listeria monocytogenes, is characterized in that specific primers represented by SEQIDNO: 1-SEQIDNO: 4 and a specific probe represented by SEQIDNO: 5 are contained in the kit, and combines the LAMP technology and the LFD detection technology, so that a detection result can be directly judged with naked eyes. The technology has the characteristics of accuracy, rapidness, convenience, efficiency, specificity and sensitiveness, is suitable for field detection of import and export quarantine, inspection for food hygiene and the like and has important significance on food safety guarantee.

Description

technical field [0001] The invention belongs to the technical field of animal hygiene and food inspection, and relates to a LAMP-LFD kit for detecting Listeria monocytogenes strains and a detection method thereof. Background technique [0002] Listeria monocytogenes (Listeria monocytogenes, LM), referred to as Listeria monocytogenes, is an important pathogen of zoonosis. The bacterium is a facultative anaerobic Gram-positive bacterium, its pH value ranges from 4.39 to 9.40, and it can grow and reproduce at 4°C, so it is very harmful to refrigerated food. Listeria monocytogenes exists widely in various environments, and it is very easy to contaminate all kinds of food. According to statistics, Listeria monocytogenes mainly contaminates dairy products, raw milk and soft cheese, livestock and poultry products, raw and cooked meat products, vegetables, etc. etc., causing foodborne listeriosis, with a fatality rate as high as 30%. In recent years, the number of food poisoning i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2565/625
Inventor 李秀梅郭恋李颖梁智选石瑜朱雅宁袁雪涛张立新王健春杨爱华尹春博王虹赵静
Owner 天津市动物疫病预防控制中心
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